Hair-binding peptides

ABSTRACT

Hair-binding peptides were isolated for their use in a variety of personal care formulations and applications. The isolation of hair-binding peptides was accomplished by enrichment using mRNA-display selection technology. Hair care compositions comprising peptide-based reagents prepared comprising the hair-binding peptides are also provided.

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/138,642 filed Dec. 18, 2008, incorporated herein byreference.

FIELD

The invention relates to the field of personal care products. Morespecifically, the invention relates to hair-binding peptides andpeptide-based hair reagents comprising hair-binding peptides.

BACKGROUND

Proteinaceous materials having strong affinity for a body surface havebeen used for targeted delivery of one or more personal care benefitagents. However, many of these materials used for targeted delivery arecomprised or derived from immunoglobulins or immunoglobulin fragments(antibodies, antibody fragments, Fab, single-chain variable fragments(scFv), and Camilidae VHH) having affinity for the target surface. Forexample, Horikoshi et al. in JP 08104614 and Igarashi et al. in U.S.Pat. No. 5,597,386 describe hair coloring agents that consist of ananti-keratin antibody covalently attached to a dye or pigment. Theantibody binds to the hair, thereby enhancing the binding of the haircoloring agent to the hair. Similarly, Kizawa et al. in JP 09003100describe an antibody that recognizes the surface layer of hair and itsuse to treat hair. A hair coloring agent consisting of that anti-hairantibody coupled to colored latex particles is also described. The useof antibodies to enhance the binding of dyes to the hair is effective inincreasing the durability of the hair coloring, but the antibodies aredifficult and expensive to produce. Terada et al. in JP 2002363026describe the use of conjugates consisting of single-chain antibodies,preferably anti-keratin, coupled to dyes, ligands, and cosmetic agentsfor skin and hair care compositions. Although single-chain antibodiesmay be prepared using genetic engineering techniques, these moleculesare expensive to prepare and may not be suitable for use in commercialpersonal care products due to their conserved structure and large size.

Non-immunoglobulin-derived scaffold proteins have also been developedfor targeted delivery of benefit agents to a target surface, such asdelivery of cosmetic agents to keratin-containing materials (See Binz,H. et al. (2005) Nature Biotechnology 23, 1257-1268 for a review ofvarious proteins used in scaffold-assisted binding). Findlay in WO00/048558 describes the use of calycin-like scaffold proteins, such asβ-lactoglobulin, which contain a binding domain for a cosmetic agent andanother binding domain that binds to at least a part of the surface of ahair fiber or skin surface, for conditioners, dyes, and perfumes.Houtzager et al. in WO 03/050283 and US 2006/0140889 also describeaffinity proteins having a defined core scaffold structure forcontrolled application of cosmetic substances. As withimmunoglobulin-like proteins, these large scaffold protein are somewhatlimited by the requirement to maintain the underlying core structure foreffective binding and are expensive to produce.

Target surface-binding peptides having strong affinity for a targetsurface have been identified and isolated from peptide libraries usingany number of biopanning techniques including, but not limited tobacterial display (Kemp, D. J.; Proc. Natl. Acad. Sci. USA 78(7):4520-4524 (1981); yeast display (Chien et al., Proc Natl Acad Sci USA88(21): 9578-82 (1991)), combinatorial solid phase peptide synthesis(U.S. Pat. No. 5,449,754; U.S. Pat. No. 5,480,971; U.S. Pat. No.5,585,275 and U.S. Pat. No. 5,639,603), phage display (U.S. Pat. No.5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571,698; and U.S.Pat. No. 5,837,500), ribosome display (U.S. Pat. No. 5,643,768; U.S.Pat. No. 5,658,754; and U.S. Pat. No. 7,074,557), and mRNA displaytechnology (PROFUSION™; U.S. Pat. No. 6,258,558; U.S. Pat. No.6,518,018; U.S. Pat. No. 6,281,344; U.S. Pat. No. 6,214,553; U.S. Pat.No. 6,261,804; U.S. Pat. No. 6,207,446; U.S. Pat. No. 6,846,655; U.S.Pat. No. 6,312,927; U.S. Pat. No. 6,602,685; U.S. Pat. No. 6,416,950;U.S. Pat. No. 6,429,300; U.S. Pat. No. 7,078,197; and U.S. Pat. No.6,436,665). Techniques to generate random peptide libraries aredescribed in Dani, M., J. of Receptor & Signal Transduction Res.,21(4):447-468 (2001). Phage display libraries are available commerciallyfrom companies such as New England BioLabs (Beverly, Mass.).

Single chain peptide-based reagents lacking a scaffold support orimmunoglobulin fold have been developed that can be used to couplebenefit agents to a target surface. Examples of target surfaces include,but not are limited to body surfaces such as hair, skin, nail, and teeth(U.S. Pat. Nos. 7,220,405; 7,309,482; and 7,285,264; U.S. PatentApplication Publication NOs. 2005/0226839; 2007/0196305; 2006/0199206;2007/0065387; 2008/0107614; 2007/0110686; and 2006/0073111; andpublished PCT applications WO2008/054746; WO2004/048399, andWO2008/073368) as well as other surfaces such as pigments andmiscellaneous print media (U.S. Patent Application Publication No.2005/0054752), and various polymers such as poly (methyl methacrylate)(U.S. Patent Application Publication No. 2007/0265431), polypropylene(U.S. Patent Application Publication No. 2007/0264720), nylon (U.S.Patent Application Publication Nos. 2007/0141629 and 2003/0185870),polytetrafluoroethylene (U.S. patent application Ser. No. 11/607,734),polyethylene (U.S. Patent Application Publication No. 2007/0141628), andpolystyrene (U.S. Patent Application Publication No. 2007/0261775).However, some single chain peptide-based reagents may lack thedurability required for certain commercial applications, especially whencoupling a particulate benefit agent to a body surface in a highlystringent matrix.

The problem to be solved is to provide additional hair-binding peptideshaving strong affinity for hair as well as peptide reagents comprisingsuch hair-binding peptides for delivery of a benefit agent to the hairsurface.

SUMMARY

The invention provides sequences of peptides that bind with highaffinity to hair. In one embodiment, a hair-binding peptide is providedhaving an amino acid sequence selected from the group consisting of: ofSEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.

In another embodiment, a peptide-based reagent is also provided, saidpeptide-based reagent comprising the general formula:

(HBP)_(n)-BA

or

[(HBP)_(m)-S]_(n)-BA

wherein;

-   -   a) HBP is at least one of the present hair-binding peptide;    -   b) BA is a benefit agent;    -   c) S is a spacer;    -   d) m ranges from 1 to about 50; and    -   e) n ranges from 1 to about 1,000.        wherein the hair-binding peptide has a sequence selected from        the group consisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13,        14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,        30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,        46, 47, 48, 49, 50, 51, and 52.

The benefit agent may be a particulate benefit agent. As such, apeptide-based reagent is provided comprising the general structure:

[(HBP)_(m)-(BABP)_(n)]_(x)

or

[[(HBP)_(m)-S_(q)]_(x)-[(BABP)_(n)-S_(r)]_(z)]_(y),

-   -   a) HBP is at least one of the present hair-binding peptides;    -   b) BABP is a benefit agent-binding peptide;    -   c) S is a molecular spacer; and    -   wherein m, n, x and z independently range from 1 to about 10, y        is from 1 to about 5, and where q and r are each independently 0        or 1, provided that both r and q may not be 0.

In a further embodiment, a hair care composition comprising an effectiveamount of at least one of the present hair-binding peptides orpeptide-based reagents is also provided.

In another embodiment, a method of delivery a benefit agent to a hairsurface is provided comprising:

a) providing a hair care composition comprising at least one of thepresent peptide-based reagents and at least one benefit agent;

b) contacting hair with the hair care composition of (a) whereby thepeptide-based reagent couples the benefit agent to hair.

The peptide-based reagents may be used to form a protective layer on ahair surface. In one embodiment, a method to form protective layer onthe surface of hair is provided comprising:

a) providing a hair-care composition comprising at least one of thepresent hair-binding peptides;

b) contacting hair with an effective amount of (a) whereby thepeptide-based reagent adheres to hair.

BRIEF DESCRIPTION OF THE BIOLOGICAL SEQUENCES

The following sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) andconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NO: 1 is the amino acid sequence of a constant N-terminal flakingregion comprising a hexa-histidine tag and a flexible linker.

SEQ ID NO: 2 is the amino acid sequence of

SEQ ID NO: 3 is the amino acid sequence of a C-terminal constant regioncomprising a flexible linker region and a C-terminal sequence optimizedfor efficient coupling to an MHA-oligonucleotide linker.

SEQ ID NOs: 4-5 are PCR primers.

SEQ ID NOs: 6-52 are hair-binding peptides biopanned against white hair.

SEQ ID NO: 53 is the amino acid sequence of the Caspase-3 cleavagesequence.

SEQ ID NOs: 54-112 are the amino acid sequence of polymer-bindingpeptides.

SEQ ID NOs: 113-116 are the amino acid sequence of various celluloseacetate-binding peptides.

SEQ ID NOs: 117-171 are the amino acid sequences of variouspigment-binding peptides.

SEQ ID NOs: 172-186 are the amino acid sequence of clay-bindingpeptides.

SEQ ID NOs: 187-212 are the amino acid sequences of calciumcarbonate-binding peptides.

SEQ ID NOs: 213-235 are the amino acid sequences of silica-bindingpeptides.

SEQ ID NOs: 236-264 are the amino acid sequences of antimicrobialpeptides.

SEQ ID NOs: 265-266 are the amino acid sequences of several peptidelinkers.

SEQ ID NOs: 267-268 are the amino acid sequences of several peptidebridges.

SEQ ID NO: 269 is the amino acid sequence of peptide CHX-W3.

SEQ ID NO: 270 is the amino acid sequence of peptide CHX-W4.

DETAILED DESCRIPTION OF THE INVENTION

Novel white hair-binding peptides having strong affinity for white hairhave been identified using mRNA-display comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 6-52. Alsoprovided are hair-binding domains formed by linking together of at leasttwo of the present hair-binding peptides In one embodiment, a firsthair-binding peptide and a second hair-binding peptide are separated byat least one linker, wherein the first and the second hair-bindingpeptide may be the same or different. In another embodiment, thespacer/linker is a peptide linker. In a further embodiment the peptidelinker is a rigid peptide linker.

The present hair-binding peptides can be used to prepare peptide-basedreagents having affinity for a hair surface. The peptide reagents maycomprise a plurality of the present hair-binding peptides coupledtogether to form a hair-binding hand. The present peptide reagents mayalso comprise at least one portion capable of being coupled to a benefitagent. In one embodiment, a portion of the peptide reagent comprises abenefit agent-binding domain.

The following definitions are used herein and should be referred to forinterpretation of the claims and the specification. Unless otherwisenoted, all U.S. patents and U.S. patent applications referenced hereinare incorporated by reference in their entirety.

As used herein, the articles “a”, “an”, and “the” preceding an elementor component of the invention are intended to be nonrestrictiveregarding the number of instances (i.e., occurrences) of the element orcomponent. Therefore “a”, “an” and “the” should be read to include oneor at least one, and the singular word form of the element or componentalso includes the plural unless the number is obviously meant to besingular.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof. The term“comprising” is intended to include embodiments encompassed by the terms“consisting essentially of” and “consisting of”. Similarly, the term“consisting essentially of” is intended to include embodimentsencompassed by the term “consisting of”.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention or employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities.

Where present, all ranges are inclusive and combinable. For example,when a range of “1 to 5” is recited, the recited range should beconstrued as including ranges “1 to 4”, “1 to 3”, “1-2”, “1-2 & 4-5”,“1-3 & 5”, and the like.

As used herein, the term “invention” or “present invention” as usedherein is a non-limiting term and is not intended to refer to any singleembodiment of the particular invention but encompasses all possibleembodiments as described in the specification and the claims.

As used herein, the terms “polypeptide” and “peptide” will be usedinterchangeably to refer to a polymer of two or more amino acids joinedtogether by a peptide bond. In one aspect, this term also includes postexpression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. Includedwithin the definition are, for example, peptides containing one or moreanalogues of an amino acid or labeled amino acids and peptidomimetics.In one embodiment, the peptides are comprised of L-amino acids.

As used herein, the term “hair” as used herein refers to human hair,eyebrows, and eyelashes. The term “hair surface” will mean the surfaceof human hair capable of binding to one of the present hair-bindingpeptides. As used herein, the term “hair-binding peptide” (HBP) refersto at least one of the present peptide sequences that bind with highaffinity to hair, such as white hair.

As used herein, the term “benefit agent' is a general term applying to acompound or substance that may be coupled with a hair-binding peptidefor application to a hair. Benefit agents typically includeconditioners, colorants, fragrances, bleaching agents, and the likealong with other substances commonly used in the personal care industry.The benefit agent may be a particulate benefit agent, such as a pigment.

As used herein, the terms “coupling” and “coupled” refer to any chemicalassociation and may include both covalent and non-covalent interactions.In one embodiment, the coupling is non-covalent. In another embodiment,the coupling is covalent.

As used herein, the term “stringency” as it is applied to the selectionof the hair-binding peptides of the present invention, refers to theconcentration of the eluting agent (usually detergent) used to elutepeptides from the hair surface. Higher concentrations of the elutingagent provide more stringent conditions.

As used herein, the terms “hair hand” and “hair-binding domain” willrefer to a single chain peptide comprising of at least two hair-bindingpeptides linked together by an optional molecular spacer, wherein theinclusion of a molecular spacer is preferred. In one embodiment, themolecular spacer is a peptide linker.

As used herein, the term “peptide-based reagent” or “peptide reagent”refers to a single chain peptide comprising at least one of the presenthair-binding peptides. In one embodiment, the peptide-based reagentcomprises two or more of the present hair-binding peptides separated bya molecular spacer. In a further embodiment, the peptide-based reagentcomprises at least two of the present hair-binding peptides separated bya peptide linker. The peptide-based reagent may also have at least oneregion that can be coupled to the benefit agent. As such, thepeptide-based reagent is used as an interfacial material to couple ahair benefit agent to the surface on human hair. The benefitagent-binding region may be comprised of at least benefit agent-bindingpeptide. In one embodiment, a benefit agent-binding domain is includedby linking together 2 or more benefit-agent binding peptides, preferablywith one or more peptide linkers.

As used herein, the term “benefit agent-binding hand” or “benefitagent-binding domain” will refer to a single chain peptide domaincomprising two or more benefit agent-binding peptides (BABPs) coupledtogether by at least one peptide linker.

As used herein, a “polymer” is a natural or synthetic compound ofusually high molecular weight consisting of repeated linked units.

As used herein, the term “pigment” means an insoluble colorant. A widevariety of organic and inorganic pigments alone or in combination may beused in the present invention. As used herein, the term “pigment lake”or “lake” refers to a pigment manufactured by precipitating a dye withan inert binder, usually a metallic salt.

As used herein, “PBP” means pigment-binding peptide. Pigment-bindingpeptides have been reported in the art (U.S. Patent Application Publ.No. 2005-0054752, U.S. Pat. No. 7,285,264) and are provided as SEQ IDNOs: 117-171. SEQ ID NOs: 142-171 bind to iron oxide-based pigments.

As used herein, “PMBP” means polymer-binding peptide. As used herein,the term “polymer-binding peptide” refers to peptide sequences that bindwith high affinity to a specified polymer (U.S. patent application Ser.No. 11/516,362). Examples include peptides that bind to poly(methylmethacrylate) (SEQ ID NOs: 54-80), polypropylene (SEQ ID NOs:81-87), polytetrafluoroethylene (SEQ ID NOs: 88-96), polyethylene(97-103), nylon (SEQ ID NOs: 104-108), and polystyrene (SEQ ID NOs:110-112).

As used herein, the term “cellulose acetate-binding peptide” is apeptide that binds with high affinity to cellulose acetate. Examples ofcellulose acetate-binding peptides are provided as SEQ ID NOs: 113-116.

As used herein, “SiBP” mean silica-binding peptide. Examples ofsilica-binding peptides are provided as SEQ ID NOs: 213-235.

As used herein, “clay-binding peptide” refers to a peptide that bindswith high affinity to clay (U.S. patent application Ser. No.11/696,380). Examples of clay-binding peptides are provided as SEQ IDNOs: 172-186.

As used herein, “calcium carbonate-binding peptide” refers to a peptidethat binds with high affinity to calcium carbonate (U.S. patentapplication Ser. No. 11/828,539). Examples of calcium carbonate-bindingpeptides are provided as SEQ ID NOs: 187-212.

As used herein, an “antimicrobial peptide” is a peptide having theability to kill microbial cell populations (U.S. patent application Ser.No. 11/516,362). Examples of antimicrobial peptides are provided as SEQID NOs: 236-264.

As used herein, the term “operably-linked” refers to the association ofnucleic acid sequences on a single nucleic acid fragment so that thefunction of one is affected by the other. For example, a promoter isoperably linked with a coding sequence when it is capable of affectingthe expression of that coding sequence (i.e., that the coding sequenceis under the transcriptional control of the promoter). In a furtherembodiment, the definition of “operably linked” may also be extended todescribe the products of chimeric genes.

As used herein, the “benefit agent” or “hair benefit agent” refers to amolecule that imparts a desired functionality or benefit when applied orcoupled to a hair surface. The present single chain peptide-basedreagents may be used to couple a benefit agent to hair. In oneembodiment, the peptide reagent is used to couple a benefit agent to ahair surface by forming a complex between the peptide-based reagent, thebenefit agent, and the hair surface. In one embodiment, thepeptide-based reagent is applied to the hair surface prior to theapplication of the benefit agent (i.e., a sequential application). Inanother embodiment, the peptide reagent and the benefit agent is appliedto the hair surface concomitantly. The benefit agent may be a peptide orthe peptide reagent (such as conditioning peptides or antimicrobialpeptides) or may be one or more molecules bound to (covalently ornon-covalently) or associated with a peptide reagent having affinity fora hair surface. The benefit agent may be a particulate benefit agent. Inone embodiment, the term “particulate benefit agent' is a general termrelating to a particulate substance, which when applied to a hairsurface provides a cosmetic or prophylactic effect. Particulate benefitagents typically include pigments, particulate conditioners, inorganicsunscreens and the like, along with other particulate substancescommonly used in the hair care industry.

The particulate benefit agent may comprise an applied coating, such as apolymeric coating or a silica coating. Examples of benefits agents mayinclude, but are not limited to conditioners for personal care products,pigments, dyes, fragrances, ultraviolet light blocking agents (i.e.,active agents in sunscreen protectants), and antimicrobial agents (e.g.,antimicrobial peptides), to name a few. In a preferred aspect, thebenefit agent is cosmetically acceptable pigment or coated pigment.

As used herein, the term “MB₅₀” refers to the concentration of thebinding peptide that gives a signal that is 50% of the maximum signalobtained in an ELISA-based binding assay (see Example 9 of U.S.Published Patent Application No. 2005-0226839; hereby incorporated byreference). The MB₅₀ provides an indication of the strength of thebinding interaction or affinity of the components of the complex. Thelower the value of MB₅₀, the stronger the interaction of the peptidewith its corresponding substrate.

As used herein, the terms “binding affinity” or “affinity” refer to thestrength of the interaction of a binding peptide (such as targetsurface-binding peptides, target surface-binding domains, and peptidereagents) with its respective substrate. The binding affinity may bereported in terms of the MB₅₀ value as determined in an ELISA-basedbinding assay or as a K_(D) (equilibrium dissociation constant) value,which may be deduced using surface plasmon resonance (SPR).

As used herein, the term “strong affinity” refers to a binding affinity,as measured as an MB₅₀ value of K_(D) value, of 10⁻⁴ M or less,preferably less than 10⁻⁵ M, more preferably less than 10⁻⁶ M, morepreferably less than 10⁻⁷ M, even more preferably less than 10⁻⁸ M, andmost preferably less than 10⁻⁹ M.

As used herein, “S” means molecular spacer. The spacer may be a peptideor non-peptide-based spacer. In one embodiment, the spacer is a “peptidespacer”. Depending upon elements with the peptide-based reagent beinglinked together the peptide spacer may also be referred to as a peptide“linker” (i.e. when linking together 2 or more target surface-bindingpeptides or “fingers” to form a binding “hand”) or a peptide “bridge”(i.e. a peptide spacer used to link/bridge a hair-binding hand to apeptide domain capable of binding to the surface of a particulatebenefit agent).

As used herein, the terms “peptide linker” will refer to a peptide usedto link together two or more target surface-binding peptides(“fingers”). In one embodiment, the peptide linker is 1 to 60 aminoacids in length, preferably 3 to 50 amino acids in length. Examples ofpeptide linkers are provided as SEQ ID NOs: 265-266.

As used herein, the term “peptide finger” will be used to refer to anindividual target surface-binding peptide, typically identified bybiopanning against a target surface.

As used herein, the term “peptide hand” will be used to refer to atarget surface-binding domain or region comprising 2 or more “fingers”coupled together using an optional peptide linker, wherein the inclusionof a peptide linker is preferred. As used herein, the term “bridge”,“peptide bridge”, and “bridging element” will refer to a linear peptideused to join a hair-binding domain (“hair-binding hand”) to a peptidedomain capable of binding to the surface of particulate benefit agent(i.e., covalent or non-covalent coupling). The peptide bridge may rangein size from 1 to 60 amino acids in length, preferably 6 to 30 aminoacids in length. Examples of peptide bridges are provided as SEQ ID NOs:267-268.

As used herein, the term “amino acid” refers to the basic structuralunit of a protein or polypeptide. The following abbreviations are usedherein to identify specific amino acids:

Three-Letter One-Letter Amino Acid Abbreviation Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Miscellaneous (or asdefined herein) Xaa X

As used herein, the term “PCR” or “polymerase chain reaction” refers toa technique used for the amplification of specific DNA segments (U.S.Pat. Nos. 4,683,195 and 4,800,159).

As used herein, the term “peptide-based” refers to an interfacialmaterial comprised of a compound pertaining to or having the nature orthe composition of the peptide class. Interfacial refers to the qualityof the peptide-based material described herein as connecting onematerial to another.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. A “foreign” gene refers to a gene not normally found in thehost organism, but that is introduced into the host organism by genetransfer. Foreign genes can comprise native genes inserted into anon-native organism, or chimeric genes.

“Synthetic genes” can be assembled from oligonucleotide building blocksthat are chemically synthesized using procedures known to those skilledin the art. These building blocks are ligated and annealed to form genesegments which are then enzymatically assembled to construct the entiregene. “Chemically synthesized”, as related to a sequence of DNA, meansthat the component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures,or automated chemical synthesis can be performed using one of a numberof commercially available machines. Accordingly, the genes can betailored for optimal gene expression based on optimization of nucleotidesequence to reflect the codon bias of the host cell. The skilled artisanappreciates the likelihood of successful gene expression if codon usageis biased towards those codons favored by the host. Determination ofpreferred codons can be based on a survey of genes derived from the hostcell where sequence information is available.

“Coding sequence” refers to a DNA sequence that encodes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within, ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

As used herein, the term “expression”, as used herein, refers to theprocess by which a gene's coded information is converted into thestructures present and operating in the cell. Expressed genes includethose that are transcribed into mRNA and then translated into proteinand those that are transcribed into RNA but not translated into protein(such as transfer and ribosomal RNAs). Expression may also refer totranslation of mRNA into a polypeptide.

As used herein, the term “transformation” refers to the transfer of anucleic acid fragment into the genome of a host organism, resulting ingenetically stable inheritance. Host organisms containing thetransformed nucleic acid fragments are referred to as “transgenic” or“recombinant” or “transformed” organisms.

As used herein, the term “host cell” refers to cell which has beentransformed or transfected, or is capable of transformation ortransfection by an exogenous polynucleotide sequence.

As used herein, the terms “plasmid”, “vector” and “cassette” refer to anextra chromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

As used herein, the term “mRNA display” is an in vitro selectiontechnique used to obtain peptides affinity that have an affinity for atarget ligand/material from libraries of diverse sequences of peptidesand proteins (U.S. Pat. No. 6,258,558). The process relies onmRNA-protein fusion molecules, which consist of peptide or proteinsequences covalently linked via their C-termini to the 3′ end of theirown mRNA (these molecules are commercially referred to as PROFUSION™molecules; Adnexus Therapeutics, Weltham, Mass.). The library ofPROFUSION™ molecules is preferably subjected to reverse transcription(i.e., transcribed into a library of DNA/RNA-protein fusion molecules)prior affinity selection. The library of fusion molecules is subjectedto repetitive rounds of in vitro selection in the presence of target(typically a solid or a material immobilized on a solid support). Aseries of washing steps are used to select the fusion moleculesexhibiting an affinity for the target material. The stringency of thewashing is adjusted to select the fusion molecules those with thehighest affinity (the affinity of the fusion molecule for the targetmaterial is attributed to the specific peptide sequence displayed).Selected fusion molecules are then subsequently subjected to PCRamplification. The end result is a pool of nucleotide sequences encodingpeptides which have an affinity for the target ligand. The process istypically repeated for several cycles and may also include mutagenesis(such as error-prone PCR) to evolve and identify proteins havingimproved affinity for the target ligand.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J. and Russell,D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y.(1984); and by Ausubel, F. M. et. al., Short Protocols in MolecularBiology, 5^(th) Ed. Current Protocols and John Wiley and Sons, Inc.,N.Y., 2002.

Binding Affinity

The present hair-binding peptides exhibit a strong affinity for hairbased on their ability to bind to hair after many rounds of selectionunder stringent conditions. The affinity of the peptide for the hair canbe expressed in terms of the dissociation constant K_(D) value or anELISA-based MB₅₀ value. K_(D) (expressed as molar concentration)corresponds to the concentration of peptide at which the binding site onthe target is half occupied, i.e., when the concentration of target withpeptide bound (bound target material) equals the concentration of targetwith no peptide bound. The smaller the dissociation constant, the moretightly bound the peptide is; for example, a peptide with a nanomolar(nM) dissociation constant binds more tightly than a peptide with amicromolar (μM) dissociation constant. In one embodiment, the presenthair-binding peptides have a K_(D) value of 10⁻⁴ M or less, preferably10⁻⁵ M or less, more preferably 10⁻⁶ M or less, even more preferably10⁻⁷ M or less, yet even more preferably 10⁻⁸ M or less, and mostpreferably 10⁻⁹ M or less.

Alternatively, one of skill in the art can also use an ELISA-based assayto calculate a relative affinity of the peptide for the target material(reported as an MB₅₀ value; see present Example 3 and co-owned U.S.Patent Application Publication 2005/022683, herein incorporated byreference). As used herein, the term “MB₅₀” refers to the concentrationof the binding peptide that gives a signal that is 50% of the maximumsignal obtained in an ELISA-based binding assay. The MB₅₀ value providesan indication of the strength of the binding interaction or affinity ofthe components of the complex. A lower MB₅₀ value is indicative of astronger interaction between the peptide with its correspondingsubstrate. In one embodiment, the MB₅₀ value (reported in terms of molarconcentration) for the hair-binding peptide is 10⁻⁴ M or less,preferably 10⁻⁶ M or less, more preferably 10⁻⁶ M or less, morepreferably 10⁻⁷ M or less, and most preferably 10⁻⁸ M or less.

mRNA-Display

The present hair-binding peptides were biopanned against white humanhair using mRNA display, an in vitro method commonly used foridentifying peptides having an affinity for a target material (U.S. Pat.No. 6,258,558). Briefly, a random library of DNA molecules is generatedwherein they encode a peptide of a desired length. The length of thepeptide within the display library is may be to be up to 200 amino acidsin length and is typically designed to range from about 7 to about 100amino acids in length. In one embodiment, the library of peptides isdesigned to be about 7 to about 60 amino acids in length, preferablyabout 7 to about 30 amino acids in length, more preferably about 15 toabout 30 amino acids in length, and most preferably about 27 amino acidsin length (i.e., a “27-mer” library). Typically, the nucleic acidmolecule encoding the peptide includes (in addition to the codingregion) appropriate 5′ and 3′ regulatory regions necessary for efficientin vitro transcription and translation. The design of the nucleic acidconstructs used for preparing the mRNA-display library is well known toone of skill in the (see WO2005/051985). The nucleic acid molecules canbe designed to optionally encode flexible linkers, cleavage sequences,fusion promoting sequences, and identification/purification tags tofacility purification and/or processing in subsequence steps.

The library of random nucleic acid fragments is transcribed in vitro toproduce an mRNA library. The mRNA is isolated and subsequently fused toa linker molecule (i.e., a puromycin-oligonucleotide linker or apuromycin derivative-oligonucleotide linker is used) using techniqueswell-known in the art (U.S. Pat. No. 6,258,558; U.S. Pat. No. 6,228,994;and Kurz et al., NAR, 28(18):e83 i-v (2000)). In a preferred embodiment,the puromycin-oligonucleotide linker comprises psoralen for rapid andfacile preparation of the mRNA-protein fusions (Kurtz et al., supra).The mRNA-puromycin fusion molecules are then translated in vitro wherebythe nascent polypeptide is fused (via the puromycin-oligonucleotidelinker) to the mRNA (PROFUSION™ molecules; Adnexus Therapeutics,Weltham, Mass.). In this way, the phenotype (peptide) is linked to thecorresponding genotype (RNA).

The mRNA-peptide fusion molecules are typically reverse transcribed intoa DNA/mRNA-protein fusion molecules prior to affinity selection. Thelibrary (often comprising up to 10¹³ different sequences) is contactedwith target ligand/material (typically an immobilized target and/or asolid surface). The selection process is carried out in an aqueousmedium wherein parameters such as time, temperature, pH, buffer, saltconcentration, and detergent concentration may be varied according thestringency of the selection strategy employed. Typically, thetemperature of the incubation period ranges from 0° C. to about 40° C.and the incubation time ranges from about 1 to about 24 hours. Theselection process is carried out in an aqueous medium wherein additionalparameters such as pH, buffer, salt concentration, and detergentconcentration may be varied according the stringency of the selectionstrategy employed.

Several washing steps are typically used to remove the non-binding/lowaffinity fusion molecules. The stringency of the washing conditions isadjusted to select those fusion molecules having the highest affinityfor the target material (such as hair). The high affinity fusionmolecules are isolated and then PCR-amplified in order to obtain thenucleic acid sequences encoding the hair-binding peptides. ThemRNA-display selection cycle is typically repeated for 3 to 10 cycles inorder to select/enrich those fusion molecules comprising peptidesequences exhibiting the highest affinity for the target material.

Error-prone PCR may optionally be incorporated into mRNA-displayselection process whereby mutants derived from a previously selectedhigh affinity sequence are used. The process is typically repeated forseveral cycles in order to obtain the peptides having improved affinityfor the target material.

Optionally, any hair-binding peptide sequence identified usingmRNA-display is verified using the free peptide. Typically, the nucleicacid molecule encoding the hair-binding peptide is cloned andrecombinantly expressed in an appropriate microbial host cell, such asE. coli. The free peptide is then isolated and assayed against thetargeted material to validate the binding affinity of the peptidesequence.

Hair-Binding Peptides

Hair-binding peptides are short, single chain peptides that bind withhigh affinity to the surface of mammalian hair, preferably human hair.The present peptides were biopanned using mRNA-display against humanwhite hair. The hair-binding peptides can be used as a benefit agent ormay be used to prepare peptide-based reagents for the delivery of abenefit agent to hair. As such, peptides having strong affinity for haircan be used to prepare peptide-based reagents capable coupling a benefitagent to the surface hair. In one embodiment, the benefit agent is aparticulate benefit agent, such as a pigment.

The present hair-binding peptides are selected from the group consistingof SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.

Single Chain Peptide-Based Reagents for Coupling a Benefit Agent to Hair

The present hair-binding peptide may be used in hair care composition tocouple a benefit agent to hair. The hair-binding peptide may also beused to form a protective layer on hair, and thus, may be considered asthe benefit agent as well.

The hair-binding peptides can be coupled directly to the benefit agentor may be coupled to the benefit agent using a molecular spacer. Assuch, a peptide-based reagent is provided comprising the generalstructure:

(HBP)_(n)-BA

or

[(HBP)_(m)-S]_(n)-BA

wherein:

-   -   a) HBP is a hair-binding peptide;    -   b) BA is a benefit agent;    -   c) S is a molecular spacer;    -   d) m ranges from 1 to about 50; and    -   e) n ranges from 1 to about 1,000.

wherein the hair-binding peptide has a sequence selected from the groupconsisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.

It may also be desirable to have multiple hair-binding peptides coupledto the benefit agent (such as a coloring agent, a condition agent, asunscreen agent or an antimicrobial agent) to enhance the interactionbetween the peptide-based reagent and the hair. Either multiple copiesof the same hair-binding peptide or a combination of differenthair-binding peptides may be used. In the case of large pigmentparticles, a large number of hair-binding peptides, i.e., up to about1,000, may be coupled to the pigment. A smaller number of hair-bindingpeptides can be coupled to the smaller dye molecules, i.e., up to about50. Therefore, in one embodiment of the present invention, thepeptide-based hair colorants are diblock compositions comprise thestructure above.

In another embodiment, the peptide-based hair colorants contain a spacer(S) separating the binding peptide from the hair coloring agent, asdescribed above. Multiple copies of the hair-binding peptide may becoupled to a single spacer molecule. In this embodiment, thepeptide-based hair colorants are triblock compositions consisting of atleast one of the present hair-binding peptides, a spacer, and a coloringagent, having the general structure [(HBP)_(m)-S]_(n)-BA, where n rangesfrom 1 to about 1,000, preferably n is 1 to about 500, and m ranges from1 to about 50, preferably m is 1 to about 10, and S is a spacer.

It should be understood that as used herein, HBP is a genericdesignation for the present hair-binding peptides. Where n or m as usedabove, is greater than 1, it is well within the scope of the inventionto provide for the situation where a series of hair binding peptides ofdifferent sequences may form a part of the composition. Additionally, itshould be understood that these structures do not necessarily representa covalent bond between the peptide, the coloring agent, and theoptional spacer. As described above, the coupling interaction betweenthe hair-binding peptide, the coloring agent, and the optional spacermay be either covalent or non-covalent.

Single Chain Peptide-Based Reagents Comprising at Least One ParticulateBenefit Agent-Binding Peptide

The hair-binding peptide may be coupled to a particulate benefit agentusing a benefit agent binding peptides. In one embodiment, the benefitagent-binding peptide is a pigment-binding peptide, a polymer-bindingpeptide, a clay-binding peptide, a calcium carbonate-binding peptide, ora silica-binding peptide.

In one embodiment, the single chain peptide-based reagent comprises afirst one portion having affinity for hair (i.e., it comprises at leastone of the present hair-binding peptides) and a second portion havingaffinity for the surface of a particulate benefit agent (i.e., comprisesat least one benefit agent-binding peptide). In one embodiment, thefirst portion comprises a plurality of hair-binding peptides withoptional peptide linkers separating the various “finger” to form ahair-binding domain (“hand”). In another embodiment, the benefitagent-binding portion comprises a plurality of benefit agent-bindingpeptides optionally separated by one or more peptide spacers for form abenefit agent-binding “hand”. As such, a peptide-based reagent is alsoprovided having the following structure:

a) a diblock peptide-based reagent having the general structure:

[(HBP)_(m)-(BABP)_(n)]_(x); or

b) a triblock peptide-based reagent having the general structure:

[[(HBP)_(m)-S_(q)]_(x)-[(BABP)_(n)-S_(r)]_(z)]_(y);

wherein:

-   -   i) HBP is a hair-binding peptide selected from the group        consisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,        16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,        32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,        48, 49, 50, 51, and 52.    -   ii) BABP is a benefit agent-binding peptide having affinity for        a hair benefit agent;    -   iii) S is a molecular spacer;    -   iv) m, n, x and z independently range from 1 to about 10;    -   v) y is from 1 to 5; and    -   vi) q an r are each independently 0 or 1, provided that both r        and q may not be 0.

The molecular spacer is preferably a peptide spacer. Peptide-spacersused to link together two or more target surface-binding peptides willbe referred to herein as “peptide linkers”. Peptide spacers that bridgetogether a hair binding hand to a benefit agent-binding hair will bereferred to herein as “peptide bridges”.

The particulate benefit agent may comprise at least one applied coating.As such, the benefit agent-binding peptide may be selected to havestrong affinity for the applied coating, such as a polymer, silica orany other cosmetically acceptable coating material. For optimal couplingof a particulate benefit agent to hair, the hair-binding hand shouldhave stronger affinity for hair than the surface on the particulatebenefit agent.

Hair Care Benefit Agents Conditioning Agents

Hair conditioning agents (HCA) as herein defined are agents that improvethe appearance, texture, and sheen of hair as well as increasing hairbody or suppleness. Hair conditioning agents are well known in the art,see for example Green et al. (WO 01/07009) and are availablecommercially from various sources. Suitable examples of hairconditioning agents include, but are not limited to, cationic polymers,such as cationized guar gum, diallyl quaternary ammonium salt/acrylamidecopolymers, quaternized polyvinylpyrrolidone and derivatives thereof,and various polyquaternium-compounds; cationic surfactants, such asstearalkonium chloride, centrimonium chloride, and Sapaminhydrochloride; fatty alcohols, such as behenyl alcohol; fatty amines,such as stearyl amine; waxes; esters; nonionic polymers, such aspolyvinylpyrrolidone, polyvinyl alcohol, and polyethylene glycol;silicones; siloxanes, such as decamethylcyclopentasiloxane; polymeremulsions, such as amodimethicone; and nanoparticles, such as silicananoparticles and polymer nanoparticles. The preferred hair conditioningagents of the present invention contain amine or hydroxyl functionalgroups to facilitate coupling to the hair-binding peptides, as describedbelow. Examples of preferred conditioning agents are octylamine (CAS No.111-86-4), stearyl amine (CAS No. 124-30-1), behenyl alcohol (CAS No.661-19-8, Cognis Corp., Cincinnati, Ohio), vinyl group terminatedsiloxanes, vinyl group terminated silicone (CAS No. 68083-19-2), vinylgroup terminated methyl vinyl siloxanes, vinyl group terminated methylvinyl silicone (CAS No. 68951-99-5), hydroxyl terminated siloxanes,hydroxyl terminated silicone (CAS No. 80801-30-5), amino-modifiedsilicone derivatives, [(aminoethyl)amino]propyl hydroxyl dimethylsiloxanes, [(aminoethyl)amino]propyl hydroxyl dimethyl silicones, andalpha-tridecyl-omega-hydroxy-poly(oxy-1,2-ethanediyl) (CAS No.24938-91-8).

The peptide-based hair conditioners may be prepared by coupling aspecific hair-binding peptide to a hair conditioning agent, eitherdirectly or via an optional spacer. The coupling interaction may be acovalent bond or a non-covalent interaction, such as hydrogen bonding,electrostatic interaction, hydrophobic interaction, or Van der Waalsinteraction. In the case of a non-covalent interaction, thepeptide-based hair conditioner may be prepared by mixing the peptidewith the conditioning agent and the optional spacer (if used) andallowing sufficient time for the interaction to occur. The unboundmaterials may be separated from the resulting peptide-based hairconditioner adduct using methods known in the art, for example, gelpermeation chromatography.

The peptide-based hair conditioners may also be prepared by covalentlyattaching a specific hair-binding peptide to a hair conditioning agent,either directly or through a spacer. Any known peptide or proteinconjugation chemistry may be used to form the peptide-based hairconditioners of the present invention. Conjugation chemistries arewell-known in the art (see for example, G. T. Hermanson, supra).Suitable coupling agents include, but are not limited to, carbodiimidecoupling agents, diacid chlorides, diisocyanates and other difunctionalcoupling reagents that are reactive toward terminal amine and/orcarboxylic acid terminal groups on the peptides and to amine, carboxylicacid, or alcohol groups on the hair conditioning agent. The preferredcoupling agents are carbodiimide coupling agents, such as1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) andN,N′-dicyclohexyl-carbodiimide (DCC), which may be used to activatecarboxylic acid groups for coupling to alcohol, and amine groups.

Additionally, it may be necessary to protect reactive amine orcarboxylic acid groups on the peptide to produce the desired structurefor the peptide-based hair conditioner. The use of protecting groups foramino acids such as t-butyloxycarbonyl (t-Boc), are well known in theart (see for example Stewart et al., supra; Bodanszky, supra; andPennington et al., supra). In some cases it may be necessary tointroduce reactive groups, such as carboxylic acid, alcohol, amine, oraldehyde groups, on the hair conditioning agent for coupling to thehair-binding peptide. These modifications may be done using routinechemistry such as oxidation, reduction and the like, which is well knownin the art.

It may also be desirable to couple the hair-binding peptide to the hairconditioning agent via a spacer. The spacer serves to separate theconditioning agent from the peptide to ensure that the agent does notinterfere with the binding of the peptide to the hair. The spacer may beany of a variety of molecules, such as alkyl chains, phenyl compounds,ethylene glycol, amides, esters and the like. Preferred spacers arehydrophilic and have a chain length from 1 to about 100 atoms, morepreferably, from 2 to about 30 atoms. Examples of preferred spacersinclude, but are not limited to ethanol amine, ethylene glycol,polyethylene with a chain length of 6 carbon atoms, polyethylene glycolwith 3 to 6 repeating units, phenoxyethanol, propanolamide, butyleneglycol, butyleneglycolamide, propyl phenyl chains, and ethyl, propyl,hexyl, steryl, cetyl, and palmitoyl alkyl chains. The spacer may becovalently attached to the peptide and the hair conditioning agent usingany of the coupling chemistries described above. In order to facilitateincorporation of the spacer, a bifunctional cross-linking agent thatcontains a spacer and reactive groups at both ends for coupling to thepeptide and the conditioning agent may be used. Suitable bifunctionalcross-linking agents are well known in the art and include, but are notlimited to diamines, such a as 1,6-diaminohexane; dialdehydes, such asglutaraldehyde; bis N-hydroxysuccinimide esters, such as ethyleneglycol-bis(succinic acid N-hydroxysuccinimide ester), disuccinimidylglutarate, disuccinimidyl suberate, and ethyleneglycol-bis(succinimidylsuccinate); diisocyanates, such ashexamethylenediisocyanate; bis oxiranes, such as 1,4 butanediyldiglycidyl ether; dicarboxylic acids, such as succinyldisalicylate; andthe like. Heterobifunctional cross-linking agents, which contain adifferent reactive group at each end, may also be used. Examples ofheterobifunctional cross-linking agents include, but are not limited tocompounds having the following structure:

where: R₁ is H or a substituent group such as —SO₃Na, —NO₂, or —Br; andR₂ is a spacer such as —CH₂CH2 (ethyl), —(CH₂)₃ (propyl), or —(CH₂)₃C₆H₅(propyl phenyl). An example of such a heterobifunctional cross-linkingagent is 3-maleimidopropionic acid N-hydroxysuccinimide ester. The Nhydroxysuccinimide ester group of these reagents reacts with amine oralcohol groups on the conditioner, while the maleimide group reacts withthiol groups present on the peptide. A thiol group may be incorporatedinto the peptide by adding a cysteine group to at least one end of thebinding peptide sequence (i.e., the C-terminus or N-terminus). Severalspacer amino acid residues, such as glycine, may be incorporated betweenthe binding peptide sequence and the terminal cysteine to separate thereacting thiol group from the binding sequence.

The spacer may be a peptide composed of any amino acid and mixturesthereof. The preferred peptide spacers are composed of the amino acidsglycine, alanine, and serine, and mixtures thereof. In addition, thepeptide spacer may contain a specific enzyme cleavage site, such as theprotease Caspase 3 site, given by SEQ ID NO: 53, which allows for theenzymatic removal of the conditioning agent from the hair. The peptidespacer may be from 1 to about 60 amino acids, preferably from 3 to about50 amino acids. These peptide spacers may be linked to the hair-bindingpeptide by any method known in the art. For example, the entire bindingpeptide-peptide spacer diblock may be prepared using the standardpeptide synthesis methods described supra. In addition, the hair-bindingpeptide and peptide spacer blocks may be combined using carbodiimidecoupling agents (G. T. Hermanson, supra), diacid chlorides,diisocyanates and other difunctional coupling reagents that are reactiveto terminal amine and/or carboxylic acid terminal groups on thepeptides. Alternatively, the entire hair binding peptide-peptide spacerdiblock may be prepared using the recombinant DNA and molecular cloningtechniques described herein. The spacer may also be a combination of apeptide spacer and an organic spacer molecule, which may be preparedusing the methods described above.

It may also be desirable to have multiple hair-binding peptides coupledto the hair conditioning agent to enhance the interaction between thepeptide-based hair conditioner and the hair. Either multiple copies ofthe same hair-binding peptide or a combination of different hair-bindingpeptides may be used. In the case of large conditioning particles (suchas particle emulsions), a large number of hair-binding peptides, such asup to about 1,000, may be coupled to the conditioning agent. A smallernumber of hair-binding peptides can be coupled to the smallerconditioner molecules, such as up to about 50. Therefore, in oneembodiment, the peptide-based reagents consisting of a hair-bindingpeptide (HBP) and a benefit agent (BA), wherein the benefit agent is ahair-conditioning agent (HCA), having the general structure(HBP)_(n)-BA, where n ranges from 1 to about 1,000, preferably from 1 toabout 50; wherein the hair-binding peptide is selected from the groupconsisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.In another embodiment, the peptide-based hair conditioners contain aspacer (S) separating the hair-binding peptide from the hairconditioning agent, as described above. Multiple copies of thehair-binding peptide may be coupled to a single spacer molecule. In thisembodiment, the peptide-based hair conditioners are triblockcompositions consisting of a hair-binding peptide, a spacer, and abenefit agent (BA) that is a hair conditioning agent (HCA), having thegeneral structure [(HBP)_(m)-S]_(n)-BA, where n ranges from 1 to about1,000, preferably n is 1 to about 50, and m ranges from 1 to about 50,preferably m is 1 to about 10.

It should be understood that as used herein, HBP is a genericdesignation referring to any one of the present hair-binding peptidesdescribed herein. Where n or m as used above, is greater than 1, it iswell within the scope of the invention to provide for the situationwhere a series of hair-binding peptides of different sequences may forma part of the composition. Additionally, it should be understood thatthese structures do not necessarily represent a covalent bond betweenthe peptide, the hair conditioning agent, and the optional spacer. Asdescribed above, the coupling interaction between the peptide, the hairconditioning agent, and the optional spacer may be either covalent ornon-covalent.

The peptide-based hair conditioners of the present invention may be usedin compositions for hair care. It should also be recognized that thehair-binding peptides themselves can serve as conditioning agents forthe treatment of hair. Hair care compositions are herein defined ascompositions for the treatment of hair, including but not limited toshampoos, conditioners, lotions, aerosols, gels, mousses, and hair dyescomprising an effective amount of a peptide-based hair conditioner or amixture of different peptide-based hair conditioners in a cosmeticallyacceptable medium. An effective amount of a peptide-based hairconditioner or hair-binding peptide for use in a hair care compositionis herein defined as a proportion of from about 0.01% to about 10%,preferably about 0.01% to about 5% by weight relative to the totalweight of the composition. Components of a cosmetically acceptablemedium for hair care compositions are described by Philippe et al. inU.S. Pat. No. 6,280,747, and by Omura et al. in U.S. Pat. No. 6,139,851and Cannell et al. in U.S. Pat. No. 6,013,250. For example, these haircare compositions can be aqueous, alcoholic or aqueous-alcoholicsolutions, the alcohol preferably being ethanol or isopropanol, in aproportion of from about 1 to about 75% by weight relative to the totalweight, for the aqueous-alcoholic solutions. Additionally, the hair carecompositions may contain one or more conventional cosmetic ordermatological additives or adjuvants including but not limited to,antioxidants, preserving agents, fillers, surfactants, UVA and/or UVBsunscreens, fragrances, thickeners, wetting agents and anionic, nonionicor amphoteric polymers, and dyes or pigments.

Peptide-Based Hair Colorants

The peptide-based hair colorants may be formed by coupling at least oneof the present hair-binding peptides (HBP) with a benefit agent thatacts as a coloring agent (C). The hair-binding peptide part of thepeptide-based hair colorant binds strongly to the hair, thus keeping thecoloring agent attached to the hair for a long lasting hair coloringeffect. The hair-binding peptides are selected from the group consistingof SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and 52.Additionally, any known hair-binding peptide may be used in combinationwith one or more of the present hair-binding peptides including, but notlimited to those described by Janssen et al. in U.S. Patent ApplicationPublication No. 2003/0152976; Janssen et al. in WO 04048399; U.S. Pat.No. 7,220,405; and U.S. patent application Ser. Nos. 11/074,473;11/359,163; and 11/251,715.

Coloring agents as herein defined are any dye, pigment, lake, and thelike that may be used to change the color of hair. In the peptide-basedhair colorants of the present invention, any known coloring agent may beused. Hair coloring agents are well known in the art (see for exampleGreen et al. supra, CFTA International Color Handbook, 2nd ed., MicellePress, England (1992) and Cosmetic Handbook, US Food and DrugAdministration, FDA/IAS Booklet (1992)), and are available commerciallyfrom various sources (for example Bayer, Pittsburgh, Pa.; Ciba-Geigy,Tarrytown, N.Y.; ICI, Bridgewater, N.J.; Sandoz, Vienna, Austria; BASF,Mount Olive, N.J.; and Hoechst, Frankfurt, Germany). Suitable haircoloring agents include, but are not limited to dyes, such as4-hydroxypropylamino-3-nitrophenol, 4-amino-3-nitrophenol,2-amino-6-chloro-4-nitrophenol, 2-nitro-paraphenylenediamine,N,N-hydroxyethyl-2-nitro-phenylenediamine, 4-nitro-indole, Henna, HCBlue 1, HC Blue 2, HC Yellow 4, HC Red 3, HC Red 5, Disperse Violet 4,Disperse Black 9, HC Blue 7, HC Blue 12, HC Yellow 2, HC Yellow 6, HCYellow 8, HC Yellow 12, HC Brown 2, D&C Yellow 1, D&C Yellow 3, D&C Blue1, Disperse Blue 3, Disperse violet 1, eosin derivatives such as D&C RedNo. 21 and halogenated fluorescein derivatives such as D&C Red No. 27,D&C Red Orange No. 5 in combination with D&C Red No. 21 and D&C OrangeNo. 10; and pigments, such as D&C Red No. 36 and D&C Orange No. 17, thecalcium lakes of D&C Red Nos. 7, 11, 31 and 34, the barium lake of D&CRed No. 12, the strontium lake of D&C Red No. 13, the aluminum lakes ofFD&C Yellow No. 5, of FD&C Yellow No. 6, of D&C Red No. 27, of D&C RedNo. 21, and of FD&C Blue No. 1, iron oxides, manganese violet, chromiumoxide, titanium dioxide, titanium dioxide nanoparticles, zinc oxide,barium oxide, ultramarine blue, bismuth citrate, and carbon blackparticles. The preferred hair coloring agents of the present inventionare D&C Yellow 1 and 3, HC Yellow 6 and 8, D&C Blue 1, HC Blue 1, HCBrown 2, HC Red 5, 2-nitro-paraphenylenediamine,N,N-hydroxyethyl-2-nitro-phenylenediamine, 4-nitro-indole, and carbonblack. In one embodiment, the coloring agent is a pigment particle, acoated pigment particle, and mixtures thereof.

Metallic and semiconductor nanoparticles may also be used as haircoloring agents due to their strong emission of light (U.S. PatentApplication Publication No. 2004/0010864 to Vic et al.). The metallicnanoparticles include, but are not limited to, particles of gold,silver, platinum, palladium, iridium, rhodium, osmium, iron, copper,cobalt, and alloys composed of these metals. An “alloy” is hereindefined as a homogeneous mixture of two or more metals. The“semiconductor nanoparticles” include, but are not limited to, particlesof cadmium selenide, cadmium sulfide, silver sulfide, cadmium sulfide,zinc oxide, zinc sulfide, zinc selenide, lead sulfide, gallium arsenide,silicon, tin oxide, iron oxide, and indium phosphide. The nanoparticlesare stabilized and made water-soluble by the use of a suitable organiccoating or monolayer. As used herein, monolayer-protected nanoparticlesare one type of stabilized nanoparticle. Methods for the preparation ofstabilized, water-soluble metal and semiconductor nanoparticles areknown in the art, and are described by Huang et al. in U.S. patentapplication Ser. No. 10/622,889. The color of the nanoparticles dependson the size of the particles. Therefore, by controlling the size of thenanoparticles, different colors may be obtained. For example, ZnS-coatedCdSe nanoparticles cover the entire visible spectrum over a particlesize range of 2 to 6 nm. Specifically, CdSe nanoparticles with a coresize of 2.3, 4.2, 4.8 and 5.5 nm emit light at the wavelength centeredon 485, 565, 590, and 625 nm, respectively. Water-soluble nanoparticlesof different sizes may be obtained from a broad size distribution ofnanoparticles using the size fractionation method described by Huang,supra. That method comprises the regulated addition of a water-miscibleorganic solvent to a solution of nanoparticles in the presence of anelectrolyte. Increasing additions of the water-miscible organic solventresult in the precipitation of nanoparticles of decreasing size. Themetallic and semiconductor nanoparticles may also serve as volumizingagents, as described above.

Of particular utility are titanium dioxide nanoparticles that not onlyserve as a colorant but additionally may serve to block harmful UVradiation. Suitable titanium dioxide nanoparticles are described in U.S.Pat. Nos. 5,451,390; 5,672,330; and 5,762,914. Titanium dioxide P25 isan example of a suitable commercial product available from Degussa.Other commercial suppliers of titanium dioxide nanoparticles includeKemira, Sachtleben, and Tayca.

The titanium dioxide nanoparticles typically have an average particlesize diameter of less than 100 nanometers (nm) as determined by dynamiclight scattering which measures the particle size distribution ofparticles in liquid suspension. The particles are typically agglomerateswhich may range from about 3 nm to about 6000 nm. Any process known inthe art can be used to prepare such particles. The process may involvevapor phase oxidation of titanium halides or solution precipitation fromsoluble titanium complexes, provided that titanium dioxide nanoparticlesare produced.

A preferred process to prepare titanium dioxide nanoparticles is byinjecting oxygen and titanium halide, preferably titanium tetrachloride,into a high-temperature reaction zone, typically ranging from 400 to2000 degrees centigrade. Under the high temperature conditions presentin the reaction zone, nanoparticles of titanium dioxide are formedhaving high surface area and a narrow size distribution. The energysource in the reactor may be any heating source such as a plasma torch.

Additionally, the coloring agent may be a colored, polymericmicrosphere. Exemplary polymeric microspheres include, but are notlimited to, microspheres of polystyrene, polymethylmethacrylate,polyvinyltoluene, styrene/butadiene copolymer, and latex. For use in theinvention, the microspheres have a diameter of about 10 nanometers toabout 2 microns. The microspheres may be colored by coupling anysuitable dye, such as those described above, to the microspheres. Thedyes may be coupled to the surface of the microsphere or adsorbed withinthe porous structure of a porous microsphere. Suitable microspheres,including undyed and dyed microspheres that are functionalized to enablecovalent attachment, are available from companies such as BangLaboratories (Fishers, Ind.).

The peptide-based hair colorants may be prepared by coupling at leastone of the present hair-binding peptides to a coloring agent, eitherdirectly or via a spacer. Any of the coupling methods described abovemay be used. It may be necessary to introduce reactive groups, such ascarboxylic acid, alcohol, amine, or aldehyde groups, on the coloringagent for coupling to the hair-binding peptide covalently. Thesemodifications may be done using routine chemistry, which is well knownin the art. For example, the surface of carbon black particles may beoxidized using nitric acid, a peroxide such as hydrogen peroxide, or aninorganic initiator such as ammonium persulfate, to generate functionalgroups. Preferably, the carbon black surface is oxidized using ammoniumpersulfate as described by Carrasco-Marin et al. (J. Chem. Soc., FaradayTrans. 93:2211-2215 (1997)). Amino functional groups may be introducedto the surface of carbon black using an organic initiator such as2,2′-Azobis(2-methylpropionamide)-dihydrochloride. The inorganicpigments and the nanoparticles may be derivatized to introducecarboxylic acid or amino functional groups in a similar manner.

Pigment-Binding Peptides

In one embodiment, the particulate benefit agent may be a pigment, acoated-pigment, or a mixture thereof. Peptide-based reagents may beprepared linking one or more of the present hair-binding peptides andone or more pigments. These bifunctional reagents may be used todelivery a pigment or coated pigment to the surface of hair (See U.S.Pat. No. 7,285,264). In one embodiment, the peptide-based reagentcomprises at least one hair-binding domain and at least onepigment-binding domains, wherein the domains linked together by anoptional spacer.

Particulate benefit agents, including pigments, may be coated with oneor more polymers known in the art. Polymer-binding peptides and/orpolymer-binding domains may be linked to one or more of the presenthair-binding peptides to form two-handed peptide reagents suitable forcoupling a polymer coated benefit agent to the surface of hair (see U.S.Patent Application Publication No. 2007-0065387). Examples of peptideshaving affinity for various polymers have also been reported.

The pigment may also be coated with silica, a material that is oftenused as a coating on pigment particles. As such, silica-binding peptidesmay be used to in the peptide-based reagent to couple hair to asilica-coated particle.

A non-limiting list of benefit agent-binding peptides, includingpeptides that bind to particulates and particulate coating materials isprovided including peptides that bind to materials such as poly(methylmethacrylate) (SEQ ID NOs. 54-80; U.S. Patent Application PublicationNo. 2007-0265431), polypropylene (SEQ ID NOs: 81-87; U.S. PatentApplication Publication No. 2007-0264720), polyethylene (SEQ ID NOs.97-103; U.S. Patent Application Publication No. 2007-0141628), polytetrafluoroethylene (SEQ ID NOs. 88-96; U.S. patent application Ser. No.11/607,734), nylon (SEQ ID NOs 104-109; U.S. Patent ApplicationPublication No. 2007-0141629), polystyrene (SEQ ID NOs. 110-112; U.S.Patent Application Publication No. 2007-0261775), cellulose acetate (SEQID NOs. 113-116; U.S. Provisional Patent Application No. 61/016,708),carbon black (SEQ ID NOs. 117-120; U.S. Patent Application PublicationNo. 2005-0054752), CROMOPHTAL® yellow (SEQ ID NOs. 121-129; U.S. PatentApplication Publication No. 2005-0054752; available from BASF Corp.,Florham Park, N.J.), SUNFAST® magenta (SEQ ID NOs. 130-132; US2005-0054752; Sun Chemical Co., Parsippany, N.J.), SUNFAST® blue (SEQ IDNOs. 133-141; U.S. Patent Application Publication No. 2005-0054752),silica (SEQ ID NOs. 213-235; co-filed, copending, and co-owned U.S.Provisional Patent Application No. 61/138,631), iron oxide (SEQ ID NOs.142-171; co-filed, copending, and co-owned US Provisional PatentApplication No. 61/138,623), clay (SEQ ID NOs. 172-186; U.S. PatentApplication Publication No. 2007-0249805), and calcium carbonate (SEQ IDNOs. 187-212; U.S. patent application Ser. No. 11/828,539).

Hair Care Compositions

The benefit agent may include any compound or material that providesbenefit to hair and typically includes, but is not limited to colorants,conditioners, sunscreen agents, antimicrobial agents, and the like.“Hair care compositions” are herein defined as compositions for thetreatment of hair including, but not limited to, shampoos, conditioners,rinses, lotions, aerosols, gels, and mousses.

An “effective amount” of the peptide-based reagent and benefit agent(combined wt %) for use in hair care compositions is a concentration ofabout 0.001% to about 20%, preferably about 0.01% to about 10% by weightrelative to the total weight of the composition. This proportion mayvary as a function of the type of hair care composition.

The concentration of the peptide-based hair reagent in relation to theconcentration of the benefit agent (such as a pigment) may need to beoptimized for best results. Additionally, a mixture of differentpeptide-based hair reagents (such as peptide-based hair colorants)having an affinity for different pigments may be used in thecomposition. The peptide-based reagents in the mixture need to be chosenso that there is no interaction between the peptides that mitigates thebeneficial effect. Suitable mixtures of peptide-based hair reagents maybe determined by one skilled in the art using routine experimentation.If a mixture of peptide-based hair coloring reagents is used in thecomposition, the total concentration of the reagents is about 0.001% toabout 20% by weight relative to the total weight of the composition.

The composition may further comprise a cosmetically acceptable mediumfor hair care compositions, examples of which are described by Philippeet al. in U.S. Pat. No. 6,280,747, and by Omura et al. in U.S. Pat. No.6,139,851 and Cannell et al. in U.S. Pat. No. 6,013,250. For example,these hair care compositions can be aqueous, alcoholic oraqueous-alcoholic solutions, the alcohol preferably being ethanol orisopropanol, in a proportion of from about 1 to about 75% by weightrelative to the total weight for the aqueous-alcoholic solutions.Additionally, the hair care compositions may contain one or moreconventional cosmetic or dermatological additives or adjuvantsincluding, but not limited to, antioxidants, preserving agents, fillers,surfactants, UVA and/or UVB sunscreens, fragrances, thickeners, wettingagents and anionic, nonionic or amphoteric polymers, and dyes.

Production of Hair-Binding Peptides

Peptides may be prepared using standard peptide synthesis methods, whichare well known in the art (see for example Stewart et al., Solid PhasePeptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984; Bodanszky,Principles of Peptide Synthesis, Springer-Verlag, New York, 1984; andPennington et al., Peptide Synthesis Protocols, Humana Press, Totowa,N.J., 1994). Additionally, many companies offer custom peptide synthesisservices.

Alternatively, peptides may be prepared using recombinant DNA andmolecular cloning techniques. Genes encoding the peptides may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts.

Preferred heterologous host cells for expression of the hair-bindingpeptides of the present invention are microbial hosts that can be foundbroadly within the fungal or bacterial families and which grow over awide range of temperature, pH values, and solvent tolerances. Becausetranscription, translation, and the protein biosynthetic apparatus arethe same irrespective of the cellular feedstock, functional genes areexpressed irrespective of carbon feedstock used to generate cellularbiomass. Examples of host strains include, but are not limited to,fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces,Pichia, Candida, Yarrowia, Hansenula, or bacterial species such asSalmonella, Bacillus, Acinetobacter, Rhodococcus, Streptomyces,Escherichia, Pseudomonas, Methylomonas, Methylobacter, Alcaligenes,Synechocystis, Anabaena, Thiobacillus, Methanobacterium, and Klebsiella.

A variety of expression systems can be used to produce the peptides ofthe present invention. Such vectors include, but are not limited to,chromosomal, episomal and virus-derived vectors, such as vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, frominsertion elements, from yeast episomes, from viruses such asbaculoviruses, retroviruses and vectors derived from combinationsthereof such as those derived from plasmid and bacteriophage geneticelements, such as cosmids and phagemids. The expression systemconstructs may contain regulatory regions that regulate as well asengender expression. In general, any system or vector suitable tomaintain, propagate or express polynucleotide or polypeptide in a hostcell may be used for expression in this regard. Microbial expressionsystems and expression vectors contain regulatory sequences that directhigh level expression of foreign proteins relative to the growth of thehost cell. Regulatory sequences are well known to those skilled in theart and examples include, but are not limited to, those which cause theexpression of a gene to be turned on or off in response to a chemical orphysical stimulus, including the presence of regulatory elements in thevector, for example, enhancer sequences. Any of these could be used toconstruct chimeric genes for production of the any of the hair-bindingpeptides of the present invention. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh level expression of the hair-binding peptides.

Vectors or cassettes useful for the transformation of suitable hostcells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, one or more selectable markers, and sequences allowingautonomous replication or chromosomal integration. Suitable vectorscomprise a region 5′ of the gene, which harbors transcriptionalinitiation controls and a region 3′ of the DNA fragment which controlstranscriptional termination. It is most preferred when both controlregions are derived from genes homologous to the transformed host cell,although it is to be understood that such control regions need not bederived from the genes native to the specific species chosen as aproduction host. Selectable marker genes provide a phenotypic trait forselection of the transformed host cells such as tetracycline orampicillin resistance in E. coli.

Initiation control regions or promoters which are useful to driveexpression of the chimeric gene in the desired host cell are numerousand familiar to those skilled in the art. Virtually any promoter capableof driving the gene is suitable for producing the binding peptides ofthe present invention including, but not limited to: CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (usefulfor expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, araB, tet, trp, P_(L), P_(R), T7, tac, and trc (usefulfor expression in Escherichia coli) as well as the amy, apr, nprpromoters and various phage promoters useful for expression in Bacillus.

Termination control regions may also be derived from various genesnative to the preferred hosts. Optionally, a termination site may beunnecessary, however, it is most preferred if included.

The vector containing the appropriate DNA sequence is typically employedto transform an appropriate host to permit the host to express thepeptide of the present invention. Cell-free translation systems can alsobe employed to produce such peptides using RNAs derived from the DNAconstructs of the present invention. Optionally it may be desired toproduce the instant gene product as a secretion product of thetransformed host. Secretion of desired proteins into the growth mediahas the advantages of simplified and less costly purificationprocedures. It is well known in the art that secretion signal sequencesare often useful in facilitating the active transport of expressibleproteins across cell membranes. The creation of a transformed hostcapable of secretion may be accomplished by the incorporation of a DNAsequence that codes for a secretion signal which is functional in theproduction host. Methods for choosing appropriate signal sequences arewell known in the art (see for example EP 546049 and WO 9324631). Thesecretion signal DNA or facilitator may be located between theexpression-controlling DNA and the instant gene or gene fragment, and inthe same reading frame with the latter.

Methods for Treating Hair

Methods are provided for treating hair with a benefit agent such aspeptide-based conditioners, colorants, sunscreen agents, andantimicrobial agents of the present invention. In another embodiment, amethod is provided for forming a protective film of peptide-basedconditioner on hair by applying one of the compositions described abovecomprising an effective amount of a peptide-based hair conditioner tothe hair and allowing the formation of the protective film. Thecompositions may be applied to hair by various means, including, but notlimited to spraying, brushing, and applying by hand. The peptide-basedconditioner composition is left in contact with hair for a period oftime sufficient to form the protective film, preferably for at leastabout 0.1 min to 60 min.

In one embodiment, a method to form a protect layer on hair is providedcomprising:

a) providing a hair-care composition comprising at least onepeptide-based reagent comprising at least one of the presenthair-binding peptides; and

b) contacting hair with the hair-care composition of (a) whereby thepeptide-based reagent adheres to hair.

A method is also provided for coloring hair by applying a hair carecomposition comprising an effective amount of a peptide-based reagentand the benefit agent to the hair by means described above. The haircare composition is allowed to contact the hair for a period of timesufficient to cause the desired effect (such as coloration,conditioning, forming a protective layer, etc.) to the hair, preferablybetween about 5 seconds to about 50 minutes, and more preferably fromabout 5 seconds to about 60 seconds, and then the hair care compositionmay be rinsed from the hair.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various uses andconditions.

The meaning of abbreviations used is as follows: “min” means minute(s),“sec” means second(s), “h” means hour(s), “μL” means microliter(s), “mL”means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm”means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s),“mM” means millimolar, “M” means molar, “mmol” means millimole(s),“μmole” means micromole(s), “g” means gram(s), “μg” means microgram(s),“mg” means milligram(s), “g” means the gravitation constant, “rpm” meansrevolution(s) per minute, “pfu” means plaque forming unit(s), “BSA”means bovine serum albumin, “ELISA” means enzyme linked immunosorbentassay, “IPTG” means isopropyl β-D-thiogalactopyranoside, “A” meansabsorbance, “A₄₅₀” means the absorbance measured at a wavelength of 450nm, “TBS” means Tris-buffered saline, “TBST” means Tris-buffered salinecontaining TWEEN® 20, “TMB” means 3,3′,5,5′-tetramethylbenzidine, “DEPC”means diethylpyrocarbonate, and “HRP” means horse radish peroxidase.

General Methods

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J. and Russell,D., Molecular Cloning: A Laboratory Manual, Third Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (2001); and bySilhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with GeneFusions, Cold Spring Harbor Laboratory Cold Press Spring Harbor, N.Y.(1984); and by Ausubel, F. M. et. al., Short Protocols in MolecularBiology, 5^(th) Ed. Current Protocols and John Wiley and Sons, Inc.,N.Y., 2002.

Materials and methods suitable for the maintenance and growth ofbacterial cultures are also well known in the art. Techniques suitablefor use in the following Examples may be found in Manual of Methods forGeneral Bacteriology, Philipp Gerhardt, R. G. E. Murray, Ralph N.Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. BriggsPhillips, eds., American Society for Microbiology, Washington, D.C.,1994, or by Thomas D. Brock in Biotechnology: A Textbook of IndustrialMicrobiology, Second Edition, Sinauer Associates, Inc., Sunderland,Mass., 1989. All reagents, restriction enzymes and materials used forthe growth and maintenance of bacterial cells were obtained from BDDiagnostic Systems (Sparks, Md.), Invitrogen (Carlsbad, Calif.), LifeTechnologies (Rockville, Md.), QIAGEN (Valencia, Calif.), Sigma-AldrichChemical Company (St. Louis, Mo.) or Pierce Chemical Co. (A division ofThermo Fisher Scientific Inc., Rockford, Ill.) unless otherwisespecified. unless otherwise specified.

Example 1 Selection of Hair Binding Peptides Using mRNA-DisplayBiopanning

The purpose of this Example was to demonstrate enrichment and isolationof hair-binding peptides using an mRNA display biopanning method.

mRNA-Display Peptide Libraries:

Methods to make libraries of DNA molecules suitable as startingmaterials for mRNA-display are well-known in the art (seeWO2005/051985). The following procedure was used to identify 27-merpeptides that have a specific affinity for hair as target material.

Briefly, a library of random nucleic acid molecules (dsDNA) eachmolecule encoding a peptide of desired length was generated. A linearpeptide library containing 81 nucleotide positions or 27 randomizedamino acid positions was used (“p27 library”). The p27 library wasdesigned to include appropriate 5′ and 3′ regions for efficient in vitrotranscription, translation, purification, and coupling to theMHA-oligonucleotide linker (MHA is3′-[α-amino-p-methoxy-hydrocinnamido]-3′-deoxy-adenosine) in theindividual molecules.

The DNA encoding the linear peptide library was designed to include a T7promoter and a tobacco mosaic virus (TMV) translation initiationsequence operably linked to the coding sequence (CDS) (Liu et al.,Methods in Enzymology, 318:268-293 (2000)). The CDS was designed toencode: (1) a constant N-terminal flaking region comprising ahexa-histidine tag followed by a flexible linker (underlined) sequence(MHHHHHHSGSSSGSGSG; SEQ ID NO: 1), (2) the randomized 27-mer linearpeptide, and (3) a constant C-terminal flanking region(TSGGSSGSSLGVASAI; SEQ ID NO: 2) comprising another flexible linkerregion (bold) and a C-terminal sequence optimized for efficient couplingto the MHA-oligonucleotide linker (double-underlined).

In Vitro Transcription

Double stranded DNA as result of the PCR reactions were transcribed intoRNA using the RiboMax Express in vitro transcription kit (PromegaMadison, Wis.). After incubation for at least 45 min at 37° C., DNase Iwas added and the incubation continued at 37° C. for additional 30minutes to degrade all template DNA. The reaction mixture was purifiedby phenol/chloroform extraction. Then free nucleotides were removed bygel filtration using G25 microspin columns (Pharmacia; Milwaukee, Wis.).Concentration of purified RNA was determined by photometry at 260 nm.

Library Preparation

Approximately 10 pmol of highly purified RNA was produced by in vitrotranscription from the p27 DNA library and purified after DNase Idigestion (by phenol/chloroform extraction and gel filtration, methodsdescribed below). The 3′-end of the p27 library RNA was modified byattachment of a MHA-linker molecule (as described ahead) and translatedin vitro by means of a rabbit reticulocyte lysate. Covalent fusionproducts between peptide and coding RNA were purified on magneticoligo(dT) beads, reverse transcribed, and again purified on a Ni-NTApurification matrix to remove uncoupled RNA and free peptides. About 8pmol of peptide-RNA-cDNA-fusions were used as input for the firstcontact with target material during selection round 1.

Chemical Coupling of RNA and MHA-oligonucleotide Linker

Purified RNA was annealed (by heat denaturation for 1 minute at 85° C.and cooling down to 25° C. for 10 minutes) with a 1.5-fold excess ofMHA-oligonucleotide linker-PEG₂A18 (5′-psoralen-UAG CGG AUG C A₁₈(PEG-9)₂ CC-MHA [nucleotides shown in italics represent2′-O-methyl-derivatives] (SEQ ID NO: 3). The covalent coupling wasinduced by radiation with UV-light (365 nm) for 15 min at roomtemperature. Aliquots of this reaction mixture before and afterirradiation with UV were analyzed on a 6%-TBE-Urea-polyacrylamidgel tocontrol the coupling efficiency (usually at least 60%).

In Vitro Translation and ³⁵S-Labelling of Peptide-RNA Fusions

Ligated RNA was translated using a rabbit reticulocyte lysate fromPromega in presence of 15 μCi ³⁵S-methionine (1000 Ci/mmole). After a 30min incubation at 30° C., KCl and MgCl₂ were added to a finalconcentration of 530 mM and 150 mM respectively in order to promoteformation of mRNA-peptide-fusions.

Oligo(dT) Purification

For the purification of peptide-RNA-fusions from translation mixturesmolecules were hybridized to magnetic oligo(dT) beads (Miltenyi Biotec;Bergisch Gladbach, Germany) in annealing buffer (100 mM Tris-HCl pH 8.0,10 mM EDTA, 1 M NaCl and 0.25% Triton X-100) for 5 min at 4° C. Beadswere separated from the mixture using MiniMACS-filtration columns(Miltenyi Biotec), repetitively washed with 100 mM Tris-HCl pH 8.0, 1 MNaCl, 0.25% Triton X-100 and finally eluted with water. A sample of thisreaction was analyzed on 4-20% Tris/glycine-SDS-PAGE; radioactive bandswere visualized using a PhosphoroImager.

Reverse Transcription (RT)

The RNAs of Oligo(dT)-purified peptide-RNA-fusions were reversetranscribed using SuperScript II Reverse Transcriptase (Invitrogen,Carlsbad, Calif.) according to the manufacturers recommendations. RTreactions contained about 1.5-fold excess of 3′-ReversePrimer. A sampleof this reaction was analyzed on 4-20% Tris/glycine-SDS-PAGE;radioactive bands were visualized using a PhosphorImager.

His-tag Purification Purification by Ni²⁺-MAC (Metal AffinityChromatography)

Reverse transcribed mRNA-peptide-fusion molecules were mixed withNi-NTA-agarose (QIAGEN; Valencia, Calif.) in HBS buffer (20 mM HEPES pH7.0, 150 mM NaCl, 0.25% Triton X-100, 100 μg/mL sheared salmon spermDNA, 1 mg/mL BSA) and incubated for 60 min at room temperature undergentle shaking. Ni-NTA was then filtrated and washed with HNT buffer (20mM HEPES pH 7.0, 150 mM NaCl, 0.025% Triton X-100) containing 5 mMimidazole. Finally peptide-RNA-cDNA-fusions were eluted with 150 mMimidazole in HNT buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 0.025% TritonX-100). A sample of this reaction was analyzed on 4-20%Tris/glycine-SDS-PAGE; radioactive bands were visualized using aPhosphorImager. BSA (final concentration 1 mg/mL) and shared salmonsperm DNA (final concentration 100 μg/mL) were added to the eluatesbefore contacting with target materials during selection step.

Selection by Binding to Target Materials and Washing

A. Incubation of Peptide-RNA-cDNA-Fusion Library with Target Material:Two different incubation buffers were used:

1. HNTriton Incubation Buffer

Purified peptide-RNA-cDNA-fusions (PROFUSION™ molecules; AdnexusTherapeutics, Waltham, Mass.) after Ni-NTA purification were incubatedfor 60 minutes at room temperature in 1 mL (final volume) of 20 mMHEPES, pH 7.4, 150 mM NaCl, 1 mg/mL BSA, 100 μg/mL shared Salmon spermDNA, 0.025% TritonX-100 in presence of DEPC-treated, blocked targetmaterial. Input activity of purified peptide-RNA-cDNA-fusions wasdetermined by scintillation measurement.

2. HNTween Incubation Buffer

For additional stringency, purified peptide-RNA-cDNA-fusions wereincubated for 60 minutes at room temperature in 1 mL (final volume) inHNTween buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 0.5% TWEEN®-20) with 1mg/mL BSA, 100 μg/mL shared Salmon sperm DNA, DEPC-treated, blockedtarget material. Input activity of purified peptide-RNA-cDNA-fusions wasdetermined by scintillation measurement.

B. Washing:

Non-binding variants were washed away by one of the following washingprocedures listed below:

-   -   Washing procedure A: used for washing the hair during selection        round 1:        -   5×5 sec. each with HNTriton buffer (20 mM Hepes, pH 7.4, 150            mM NaCl, 0.025% Triton-X100)        -   1×5 sec 150 mM NaCl (for buffer removal before elution with            KOH)    -   Washing procedure B: used for washing of hairs during selection        round 2-11, and 13-control for white hair:        -   1×5 sec. each with HNTween buffer (20 mM Hepes, pH 7.4, 150            mM NaCl, 0.5% Tween-20)        -   2×5 min. with 10% shampoo in HNTriton buffer        -   2×5 sec with HNTween buffer        -   1×5 sec 150 mM NaCl (for buffer removal before elution with            KOH)    -   Washing procedure C : used for washing of hair during selection        rounds 11b, 12b, and 13b for white hair.        -   2×5 sec. each with HNTween buffer (20 mM Hepes, pH 7.4, 150            mM NaCl, 0.5% Tween-20)        -   1×5 min. with 10% shampoo in HNTriton buffer        -   1×5 sec with HNTween buffer including tube change        -   4×30 min with 10% shampoo in HNTriton buffer        -   3×5 sec with HNTween buffer; 1 tube change during the third            wash        -   1×5 sec 150 mM NaCl (for buffer removal before elution with            KOH)

The shampoo used in the above washing procedures was a commerciallyavailable hair shampoo having the following composition:

Water 51% Ammonium lauryl sulfate 20% Sodium lauryl ether sulfate 15%Cocamidopropyl betaine 7% Cocamide MEA 2.5% Miscellaneous minorcomponents** ~4.5% **(e.g. various pH adjusters, preservatives,vitamins, chelating agents, dispersants, lubricants, fragrances, anddyes)

Comment on Incubation and Washing Conditions:

Normally during mRNA display selections a low detergent concentration ischosen to have low stringent conditions during up to 6 rounds ofselection by keeping the detergent concentration at 0.025% Triton-X100.However, a higher stringency for the target material was applied fromthe beginning during incubation and washing (see washing procedures).The applied high concentrations of TWEEN®-20 and shampoo are close tothe so called “critical micelle concentration” (CMC) allowing theformation of small micelles which might contain more than onepeptide-RNA-cDNA-fusion. Since CMC driven aggregation ofpeptide-RNA-cDNA-fusions are critical for successful selections, higherconcentrations of the detergents described above were not used.

cDNA Elution:

cDNAs of binding variants were eluted by incubation of target materialin 50 μL of 100 mM KOH at 60° C. for 30 minutes. After centrifugation,supernatant was removed from target material and transferred into afresh tube. KOH eluates were subsequently neutralized by addition of 1μL of 1 M Tris/HCl, pH 7.0 and 3.8 μL of 1 M HCl (per 50 μL 100 mM KOH).

Polymerase Chain Reaction (PCR):

After elution in KOH and neutralization, the recovered cDNAs wereamplified by quantitative PCR with increasing numbers of amplificationcycles (12, 15, 18, 21, 24 and 27 cycles). Products were subsequentlyanalyzed by agarose gel electrophoresis over 2% agarose gels. Optimizedconditions (minimal cycle number to get good enrichment of DNA ofcorrect length) were then applied for a preparative PCR reaction andcontrolled again by agarose gel electrophoresis.

Analytical and preparative PCR reactions were performed in presence of10 mM Tris-HCl (pH 8.8 at 25° C.), 50 mM KCl, 0.08% Nonidet P40, 2 mMMgCl₂, 2.5 mM dNTPs, 1 μM of each forward and reverse primer(5′-TAATACGACTCATAGGGACAATTACTATTTACAA TTACAATG-3′; SEQ ID NO: 4) and(5′-AATTAAATAGCGGATGCTACACCAAGACTAGAACCGCTG-3′; SEQ ID NO: 5), 1/5volume of neutralized cDNA eluate and 0.05 U/μL Taq polymerase(Promega). Temperature program of PCR reaction is given below: Initialdenaturation: 90 sec at 94° C.; cycling: 15 sec at 94° C.(denaturation), 20 sec at 60° C. (annealing), 30 sec at 72° C.(extension); post treatment: 3 min at 72° C. (post-treatment); hold at4° C.

Enrichment of cDNA-RNA-Peptide Fusion Molecules Binding to White Hair

Initial selection was conducted using stringent washing conditions.Thirteen rounds of selection were conducted and the relative binding ofradioactively labeled cDNA-RNA-peptide fusion molecules to the whitehair target material was measured. The amount of target used per roundwas 4 hairs stuck together with glue in a brush-like structure.

Rounds 1 of selection used washing procedure A. Rounds 2-9 and13-control used washing procedure B. Rounds 10, 12, and 13 used washingprocedure C. Round 11 used washing procedure D. The relative amount ofenrichment (reported as percent enrichment of binding molecules relativeto their respective input signals [activity of cDNA-RNA-peptide fusionsbefore contacting with the target material]) is provided in Table 1.

TABLE 1 % Enrichment of cDNA- RNA-peptide fusion Selection IncubationWashing molecules having an Round Buffer Procedure affinity for whitehair 1 1 A 0.0 2 1 B 0.070 3 1 B 0.140 4 1 B 0.459 5 1 B 3.536 6 1 B0.513   7^(a) 1 B 2.928^(a) 8 2 B 0.890 9 2 B 0.310 10  2 B 1.617 11  2B 0.821 11b 2 C 0.187 12b 2 C 0.222   13b^(a) 2 C 0.108^(a) 13-control 2B 0.824 ^(a)= enriched binder sequences of respective round analyzed

Sequencing of 27-mer White Hair-Binding Peptides

The cDNA molecules from the enriched pool of white hair-binding fusionmolecules were isolated and PCR amplified as described above. Thesequences of the DNA molecules encoding the white hair-binding peptideswere determined and are provided in Tables 2 (Round 7), and Table 3(Round 13).

TABLE 2 Enriched White Hair-binding Peptides After Round 7. SEQ SampleID No. Amino Acid Sequence NO:  1 RPGRRIRPVLKATEWLLVSISKLLWGM  6  2LCRNRRGLYRSLLSSAKRGVSSFLWWT  7  3, 28 VKKAKAQRVRGISLWNFVCHVSRGLWT  8  5LGMLMRRMIKMGKLLSALGLWYRPRVS  9  6, 29, 30 RRMRIRWLSLLSWLVSGLNRRTHLGRP 10 7, 24 KCGQVSRLGWNRNAWWKFKVLSSVWGW 11  8 GRRSLHLRTGIMSVRPFRYLCTLQRMW 12 9 RNAHGRRSRNHMWWGLARLALIGFLTV 13 10 GRTRQECRRTTWWNRVFVTVKRFTSGG 14 11RILRGVRRVLGPPMLWPVRFWNWMVKA 15 12 VWSWLKRAMYTKFLLGVSRYVFRT 16 13VARWRRILSRAWNASRLMAWVMWHKSH 17 14 VIWRRPGCLKSFGRPWFVVFRKVGLHF 18 15GRSHVFRVLKSVMWLFKKLAVLSGRAA 19 16 RPRWKGWSWIKLGLQVMHGLPRLLGNR 20 17GSRKRLRSNLVTYLRIGHSLLSVIRRA 21 18 VVASRRKQRMAASKIYDWYRCVYDWFT 22 19GRKRNASLWSRVHVLRWFPLLTWALLQ 23 20 RKVRWAKSLRLVSIFWRLWKSNFTTYE 24 21RSMVHRFRLGVRRLAWGLAIRGLRIMM 25 22 FHKRRATLRSSLISKVMGLVLNKLMGS 26 23NSNKHRMIPMRRWTWLAITTLRAFRWA 27 25 AVWKKTRIWRRIPMYRFVGYLVRMFTS 28 26VLTRLGRVARKAWDKWWCRFKVSAASH 29 27 RNWVRAMSKVWPSWRLMWWLGTLGSNP 30

TABLE 3 Enriched White Hair-binding Peptides After Round 13. SEQ IDSample No. Amino Acid Sequence NO:  1, 7 SIKKLVMRMLVGLLVRHRVKHALLNHW 31 2, 14, 24, VLRRVHRLLTLVSVARKATRSVYQWLF 32 31, 33, 35  3RLALKTRRSVQTARLMLNLWHVLGNWS 33  4, 5 RRSMSLRMLTRGIKWSAMAYLASRWIL 34 6, 12 SLRRRWTTFGKILRRWTLPTAMLLVMM 35  9, 17, 25,RLALKTRRSVQTVRLMLNLWHVLGNWS 36 30, 40 10 RTRRSSRTATIYKLQLLMHSWKLLHLL 3711 RIVRGKKRLNLKRDLVRLLWQATWWRF 38 13 LRRLVRRLRLLEFMWTGLSSVSKNIFG 3915, 18 FGHLRRTSTKWYKALLRNSLLWGIWRI 40 16 LGRKILRRIAKTVWTRLSERFMSNIWL 4119 FRFRVPKLRLGQLWWLTWPLLKWTQNA 42 20 RRWRMLLHDMLLGMTIKVFRKVKFRYL 43 22RQTSWVHRALRWVRIGTAISEGILRGM 44 23 ARLRKKAMIRVLGKTAMWWLGTWMGHA 45 26RMTFSKRVLASLVIKPLISSTWAWILN 46 27 ALRRVHRLLTLVSVARKATRSVYQWLF 47 28QQGAGLLKQLVARKFLSWGLNTLIT 48 32 LRQVHRAVRRGRLIHKVVTWGWHWFVT 49 34ERLRAARFQRKIMFVLARLWLGPIWHR 50 36 RRVIRHLWKRLILSGSNVMLAWLLKGS 51 38VGKRIHLARVFWRTWHMGSVFMRFLKA 52

Example 2 Determination of Hair-Binding Affinity

The purpose of this Example was to determine the affinity ofhair-binding peptides for hair surfaces, measured as MB₅₀ values, usingan ELISA assay. Several hair-binding peptides from Example 1 were usedto confirm that the selection process used in Example 1 producedhair-binding peptides having strong affinity for hair.

The peptides were synthesized using standard solid phage synthesismethod and were biotinylated by adding a biotinylated lysine residue atthe C-terminus of the amino acid binding sequence for detectionpurposes. The peptides tested were CXH-W3 (Sample ID Nos. 4 and 5 fromTable 3; SEQ ID NO: 34) and CXH-W4 (Sample ID NOs: 6 and 12 from Table3; SEQ ID NO: 35).

The MB₅₀ measurements of the biotinylated peptides binding to hair weredone using the natural white hair bundles (piedmont white human hairswere obtained from International Hair Importers and Products (Bellerose,N.Y.). The hair samples were assembled in bundles consisting of 100hairs about 1 cm long which were bundled together using narrow tape atone end. The hair bundles were incubated in SUPERBLOCK® blocking buffer(Pierce Chemical) for 1 hour at room temperature (˜22° C.), followed by3 washes with TBST (TBS in 0.05% TWEEN® 20). Peptide binding bufferconsisting of various concentrations of biotinylated peptide in TBST and1 mg/mL BSA was added to the hair bundles and incubated for 1 hour atroom temperature, followed by 6 TBST washes. Then, thestreptavidin-horseradish peroxidase (HRP) conjugate (Pierce ChemicalCo., Rockford, Ill.) was added to each well (1.0 μg per well), andincubated for 1 h at room temperature, followed by 6 times of washeswith TBST. All hair bundles were transferred to new tubes and then thecolor development (3,3′,5,5′-tetramethylbenzidine (TMB) was thesubstrate) and the absorbance measurements were performed following themanufacturer's protocol. The results were plotted as A₄₅₀ versus theconcentration of peptide using GraphPad Prism 4.0 (GraphPad Software,Inc., San Diego, Calif.). The MB₅₀ values were calculated from Scatchardplots and are shown Table 4.

TABLE 4 Summary of MB₅₀ Values for Selected Hair-Binding PeptidesPeptide Peptide Sequence ID (SEQ ID NO:) MB₅₀ (M) CXH-W3RRSMSLRMLTRGIKWSAMAYLASRWIL- 7.2 × 10⁻⁸ K(biotin)-NH₂ (SEQ ID NO: 269)CXH-W4 SLRRRWTTFGKILRRWTLPTAMLLVMM- 2.1 × 10⁻⁸ K(biotin)-NH₂(SEQ ID NO: 270)

1-15. (canceled)
 16. A hair-binding peptide selected from the groupconsisting of SEQ ID NOs: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, and
 52. 17.A peptide-based hair care reagent having the general structure:(HBP)_(n)-BAor[(HBP)_(m)-S]_(n)-BA wherein: a) HBP is a hair-binding peptide; b) BA isa benefit agent; c) S is a molecular spacer; d) m ranges from 1 to about50; and e) n ranges from 1 to about 1,000. wherein the hair-bindingpeptide has a sequence selected from the group consisting of SEQ ID NOs:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, and
 52. 18. The peptide-based reagent ofclaim 17 wherein the benefit agent is selected from the group consistingof colorants, conditioning agents, and antimicrobial agents.
 19. Thepeptide-based reagent of claim 18 wherein the colorant is a dye, a lake,or a pigment.
 20. The peptide-based hair care reagent of claim 18wherein the conditioning agent is selected from the group consisting ofcationic polymers, cationic surfactants; fatty alcohols, fatty amines,nonionic polymers, silicones, siloxanes, polymer emulsions, andnanoparticles.
 21. The peptide-based reagent of claim 17 wherein thehair-binding peptide is generated by mRNA display.
 22. A peptide-basedreagent having the general structure: a) a diblock peptide-based reagenthaving the general structure:[(HBP)_(m)-(BABP)_(n)]_(x); or b) a triblock peptide-based reagenthaving the general structure:[[(HBP)_(m)-S_(q)]_(x)-[(BABP)_(n)-S_(r)]_(z)]_(z); wherein: i) HBP is ahair-binding peptide selected from the group consisting of SEQ ID NOs:6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, and
 52. ii) BABP is a benefitagent-binding peptide having affinity for a hair benefit agent; iii) Sis a molecular spacer; iv) m, n, x and z independently range from 1 toabout 10; v) y is from 1 to 5; and vi) q an r are each independently 0or 1, provided that both r and q may not be
 0. 23. The peptide-basedreagent of claim 22 wherein the benefit agent-binding peptide isselected from the group consisting of pigment-binding peptides,polymer-binding peptides, clay-binding peptides, calciumcarbonate-binding peptides, and silica-binding peptides
 24. Thepeptide-based reagent of claim 22 wherein the hair-binding peptide isgenerated by mRNA display.
 25. The peptide-based reagent of claim 22wherein benefit agent is a particulate benefit agent.
 26. Thepeptide-based reagent of claim 25 wherein the benefit agent is selectedfrom the group consisting of pigments, conditioners, and sunscreenagents.
 27. A hair-care composition comprising the hair-binding peptideof claim
 16. 28. A hair-care composition comprising the peptide-basedreagent of claim 17 or claim
 22. 29. A method for applying a benefitagent, colorant, or conditioner to hair comprising: a) providing thehair-care composition of claim 28; and b) contacting hair with the haircare composition of (a) whereby the peptide-based reagent couples thebenefit agent to hair.
 30. A method for form a protective layer on hairis provided comprising: a) providing the hair-care composition of claim28; and b) contact hair with the hair-care composition of (a) wherebythe peptide-based reagent adheres to hair.